Role of NAXT nitrate excretion transporters in stomatal aperture control and plant tolerance to salinity and drought. – NAXTRESS

The project started with the selection of those NAXT members found to be specifically expressed in stomata, thereby possibly involved in the control of transpirational water losses under conditions of stress (mainly salt). Localization of expression in stomata was then confirmed using confocal microscopy on several Arabidopsis transgenic lines expressing transcriptional and translational fusions with reporter genes. Functional characterization of NAXT members was performed at the whole plant, integrated level using knock-out mutants under different growth conditions in order to identify in which environmental contexts do NAXT members provide adaptive advantages to plants equipped with functional alleles. Expression of some NAXT members in heterologous system (Lactococcus lactis) completed their functional characterizations at the cellular level.

NAXT2 has been identified as an efflux transporter for NO3-, located in roots and involved in NO3- translocation towards the leaves. Contrary to the expression of NRT1.5 (major transporter for NO3- under standard conditions, located in the root stele), expression of NAXT2 was not repressed under salt stress. NAXT2 helps limit the decrease of leaf NO3- content induced by Na+ in the root medium. It mitigates the negative impact of salt stress on plant growth. It also participates in the control of water losses when the plant experiences salt stress. In this latter case, the protein directly acts in the guard cells which control stomatal aperture through which water vapor leaves the plant. Within the same protein family, NAXT1 and NAXT3 likely play a role in stomatal closure under salt stress too. Original results have also been obtained regarding acquisition of stomatal sensitivity to atmospheric conditions during leaf ontogeny.

Scientific results obtained during this project open new avenues for selecting plants able to better withstand conditions of moderate salt stress. They also adress strengthen the importance of nitrate nutrition in plant tolerance to salt stress.
Beyond this scientific outcomes about the roles of some NAXT members, the project reinforced the connections between the participants (1 post-doc funded by ANR was co-supervised and 2 PhD students funded by other sources were also co-supervised). Thematic approaches of each partner have been combined to reveal new mechanisms of plant tolerance to salt stress, thereby strengthening the leader position of all 3 partners in the domain of plant tolerance to abiotic stresses. The partnership goes beyond the framework of this project. Two of the partners committed in the project NAXTRESS carry on with collaborations within a new, recently funded, ANR project, dedicated to analysis of potassium fluxes in grapes. Another collaboration with the third partner has been initiated using similar techniques as those developed during the project NAXTRESS. This should result in a major publication about plant tolerance to high temperature.

Original results obtained in this project have been published in first rank, international journals (one publication under revision for Plant Cell and another one published in Curr. Biol.) and presented in renowned international meetings (Gordon Conference).

Nitrate is the major mineral anion in cultivated plants and is essential for their N-nutrition. Although its uptake from the medium is energetically costly, numerous abiotic stresses (mechanic shocks, salinity, medium acidification...) induce a net root NO3- excretion into the medium. The physiological significance of this excretion remains obscure. It is nevertheless aknowledged that anion excretion is a major determinant for the control of electric polarization (and related signalization pathways) and osmotic potential in plant cells. For instance in guard cells, anion excretion leads in fine to stomata closure.
The molecular knowledge of transport systems responsible for cellular efflux of NO3- in plants is scarce. One of the 3 partners of this project identified NAXT1 (NitrAte eXcretion Transporter 1), the first NO3- efflux transporter at the plasma membrane of plant cells (Segonzac et al., 2007, Plant Cell). NAXT1 is responsible for the massive root NO3- excretion to the external medium, triggered by acidification stresses. In Arabidopsis, the NAXT family encompasses 7 closely related genes whose expression is detected in roots at different levels. Upon salt stress, NAXT2 (another member) was found to be involved in NO3- excretion in the root stele and its translocation to shoots.
Recently, one of the partners recently observed that NAXT1 and NAXT2 are also expressed in leaves, at the level of guard cells that constitute stomata. A first stomata-related phenotype was uncovered in salt stress conditions for a NAXT2 KO mutant (enhanced leaf transpiration rate compared to wild type plants). Also, NAXT1 and NAXT2 gene expression is regulated by the stress hormone ABA and salt stress. These first observations are of importance because stomata constitute an essential control point for plant tolerance to agronomicaly important stresses such as salinity and drought. For these reasons, it is proposed in the present project to study the role of NAXT transporters in stomatal movements in Arabidopsis, with the aim to uncover new plant tolerance factors to these stresses.
In line with this proposal, the composition of our consortium allows to associate recognized expertise in (1) electrophysiological analyses of transport systems [Montpellier, BPMP], (2) anion channel and stomatal activity [Cadarache, IBEB-LEMS], and (3) Integrative biology of plants under environmental constraints [Montpellier, LEPSE].
First, the expression of NAXT genes will be searched for in guard cell RNAs extracted from plants submitted or not to treatments of interest (salt and water stress, ABA). Transport properties of selected NAXT members (expressed in stomata) will be characterized electrophysiologically after expression in Xenopus oocytes, and their expression profile in stomata will be documented at the gene and protein levels (in NAXT:GFP plants) in response to stresses and ABA. Then, NAXT underexpressing mutants will be phenotyped in response to stresses on their stomatal activity (aperture measurements, electrophysiology in situ) and by an integrative approach (tolerance to stresses in terms of growth/development, leaf transpiration rate) using a phenotyping platform (Phenopsis) developped by one of the partners.
Beyond the deciphering of the biological role of the NAXT family, it is anticipated that this project will allow a better understanding of the beneficial role of nitrate supply observed at an agronomic level to plants under major abiotic stresses limiting production and thereby, to uncover new determinants of their tolerance.